CYP genes are duplicated in Drosophila suzukii and carry transposable elements

Gene duplication and transposable element (TE) insertions are key drivers of genome evolution and have been linked to increased insecticide resistance in insects. The aim of this study was to characterize the distribution and potential impact of TEs within and near cytochrome P450 monooxygenase (CYP) genes in the invasive pest Drosophila suzukii compared with the non-pest Drosophila melanogaster. We performed in silico analyses of the CYP gene repertoire and genome architecture in D. suzukii and D. melanogaster. We identified ten duplicated CYP genes that are exclusive to D. suzukii and absent from its closely related species D. melanogaster. These duplications are enriched with TE fragments, predominantly Helitrons. Thirty-six percent of TE sequences within CYP genes and their flanking regions carry putative transcription-factor binding sites in D. suzukii, indicating a possible role in gene regulation. Building on the proposed model of Helitron-mediated exon shuffling, our findings suggest that these elements contribute to gene rearrangement, thereby potentially enhancing functional diversity. At the genome level, D. suzukii harbors a higher overall TE content than D. melanogaster, with a relative enrichment within CYP genes. The increased TE content may have enhanced genomic plasticity, thereby facilitating the species’ invasive success, rapid population growth, and ability to adapt to diverse habitats, such as native environments and agricultural fields.

Introduction

Cytochrome P450 monooxygenases (CYPs) are a diverse group of isoenzymes that play a fundamental role in the metabolism of endogenous and exogenous compounds (Feyereisen, 2005). Belonging to an ancient and widespread gene family, CYPs are present in virtually all living organisms (Feyereisen, 2005). In insects, they are critical for detoxification processes, contributing to the metabolism of both natural plant allelochemicals and synthetic insecticides (Scott, 1999; Feyereisen, 2005; Li et al., 2007). Variation in CYP gene expression and copy number has often been linked to the development of metabolic resistance to insecticides in arthropod pest species (Hu et al., 2025).

Resistance to insecticides serves as a valuable model for studying evolutionary phenomena because the selective agent (insecticide) is well understood, and the response to selection (resistance) is typically rapid (McKenzie and Batterham, 1994). For instance, insecticide resistance has been associated with overexpression of CYP6p3 in Anopheles gambiae (Müller et al., 2008), and CYP6bq9 in the brain of Tribolium castaneum (Zhu et al., 2010). In the aphid Myzus persicae, resistance is mediated by duplication of CYP6cy3 and CYP6g1 (Puinean et al., 2010), whereas in Drosophila melanogaster it involves both duplication and overexpression of CYP6g1 (Daborn et al., 2002; Harrop et al., 2014).

In addition to gene duplication and regulatory changes, the activity of transposable elements (TEs) has emerged as a key mechanism in modulating CYP gene expression and driving the evolution of insecticide resistance. TEs are repetitive DNA sequences capable of moving within and between genomes and this mobility can affect genome structure and function, leading to phenotypic changes through altered gene expression and increases in genome size (Elliott and Gregory, 2015). In Drosophila, TEs - including insertions and TE-derived fragments - have been shown to shape CYP genes regulation and contribute to insecticide resistance (Daborn et al., 2002; Catania et al., 2004; Schlenke and Begun, 2004; Bogwitz et al., 2005; Marsano et al., 2005; Chung et al., 2007; Carareto et al., 2013). For instance, overexpression of CYP6g1 in D. melanogaster is associated with the insertion of the Accord retroelement upstream of the gene (Daborn et al., 2002), while in D. simulans, the ortholog is overexpressed due to insertion of the DOC element in its flanking region (Schlenke and Begun, 2004). Moreover, in D. melanogaster, insertion of the Bari-1 element at the 3’ end of CYP12a4 enhances gene expression (Bogwitz et al., 2005; Marsano et al., 2005). Among these, Helitrons—rolling-circle DNA transposons capable of capturing and reshuffling host gene fragments—represent a particularly dynamic family whose role in CYP evolution is explored in detail in this study.

Drosophila species are best known as model organisms in genetics and evolutionary biology, but a few have become significant agricultural pests. Among them, Drosophila suzukii (Matsumura, 1931) is notable as one of the most damaging fruit crop pests worldwide (Walsh et al., 2011; Asplen et al., 2015). Native to Japan, D. suzukii has spread throughout Asia; North America (Walsh et al., 2011; Asplen et al., 2015; Cini et al., 2012; Rota-Stabelli et al., 2013); Europe (Kaneshiro, 1983; Leblanc et al., 2009); and South America, where our group first reported its occurrence (Deprá et al., 2014). Females possess a serrated ovipositor that allows them to lay eggs in healthy fruits rather than decaying ones (Walsh et al., 2011; Lee et al., 2015). The resulting perforations provide entry points for pathogens, leading to economic losses of up to 80% in fruit production, mostly berries (Dreves et al., 2009; Hauser, 2011; Escudero et al., 2012), and releasing volatile compounds (Abraham et al., 2015) that attract other drosophilid species (Timmeren and Isaacs, 2013; Joshi et al., 2014; Lasa and Tadeo, 2015). Once established, D. suzukii is extremely difficult to eradicate, resulting in increased production costs due to the need for constant monitoring, intensive management, greater insecticide use, and post-harvest fruit sorting. Given its invasive success, broad ecological tolerance, and heavy exposure to insecticides, D. suzukii provides an ideal model to investigate how gene duplication and transposable elements contribute to genomic plasticity and adaptive evolution.

Understanding the genetic basis of Drosophila suzukii’s adaptability and resistance to control measures is essential for developing effective management strategies. The sequenced genome of this species (Chiu et al., 2013) contains 76 annotated CYP genes (SpottedWingFlyBase, Annotation Release v1), compared with 99 in D. melanogaster (FlyBase, Release v3). Since CYPs are among the major metabolic systems in insects capable of mediating resistance to all major classes of insecticides (Scott, 1999; Li et al., 2007), examining these genes—along with their associated TEs and potential regulatory effects—can provide valuable insights into the genetic and molecular mechanisms underlying insecticide resistance and the species’ invasive success.

To address this issue, the main aim of this study was to compare the CYP gene repertoire of D. suzukii and D. melanogaster, focusing on gene structure, the occurrence and distribution of TE insertions within or near these genes, to explore their potential influence on gene structure and TE-derived regulatory elements. We hypothesized that transposable elements (TEs) may contribute to regulatory diversification, potentially playing a key role in the adaptive success and insecticide resistance of D. suzukii.

Materials and methods

In silico analysis of CYP genes

CYP gene sequences and structures were retrieved directly from the curated genome annotations available in the Gbrowser databases (SpottedWingFlyBase, SpottedWingFlyBase, 2013, and FlyBase, FlyBase, 1993), using Annotation Release v1 for D. suzukii and Annotation Release v3 for D. melanogaster (Supplementary Tables S1-S4). Only protein-coding CYP genes were included in the analysis. Because these genes were obtained from assembled annotations rather than from de novo searches, no overlapping or ambiguous hits were encountered. As transposable elements (TEs) in flanking regions can provide novel transcriptional regulatory signals, we extracted 10 kb upstream (5′ flanking region) and 10 kb downstream (3′ flanking region) of each CYP gene based on the annotated gene coordinates. The retrieved sequences were visually inspected in Gbrowser to compare genomic features between species.

Gene length comparisons between D. suzukii and D. melanogaster were performed and visualized in R using the genoPlotR package (Guy et al., 2010). In this package, each orthologous gene is plotted side-by-side to allow direct structural comparison, enabling visualization of local synteny among the species such as exon–intron organization, total gene length, and the positions of TE insertions. All graphical outputs were refined in Inkscape v0.92.1¹.

For phylogenetic context, we incorporated the maximum-likelihood phylogeny generated by Chiu et al. (2013) into our comparative analyses. We further expanded these analyses to include orthologous CYP genes from two sister species of D. suzukii, Drosophila biarmipes and Drosophila takahashii (Annotation Release 101 for both species), and screening these orthologs for TE insertions (Supplementary Tables S5, S6) using the same RepeatMasker-based pipeline described below to ensure robust cross-species comparisons.

In silico analysis of transposons

To detect the presence of TEs associated with CYP genes, we analyzed each gene sequence and, separately, 10 kb of its 5′ and 3′ flanking regions using the RepeatMasker web server (RepeatMasker Open-4.0 Software, 1996). Searches were conducted with the parameters: crossmatch, fruit fly, and GC-level–based matrix. TE classification was assigned according to the highest-scoring match using the Drosophila reference library from Repbase (Jurka et al., 2005). Because the “fruit fly” RepeatMasker library is optimized for D. melanogaster, it may therefore underestimate lineage-specific or recently diverged TE families in D. suzukii.

Based on the RepeatMasker output, we classified TE fragments according to their genomic location relative to CYP genes as: (i) intronic, (ii) 5′ flanking (within 10 kb upstream of the transcription start site), or (iii) 3′ flanking (within 10 kb downstream of the transcription termination site). TE sequences located within CYP genes and 10 kb of its flanking regions were further analyzed to identify putative transcription-factor binding sites (TFBS). Strand-specific predictions were performed using the ConSite web server (ConSite Software, 2004) with the JASPAR CORE Insecta database (Bryne et al., 2008) for D. melanogaster, applying a 90% TFBS cutoff score, following the methodology of Carareto et al. (2013).

To evaluate whether the number of TE insertions in CYP genes was proportional to the overall genomic TE composition of the studied species, Illumina reads were obtained from the Sequence Read Archive (SRA): D. suzukii – SRR942805 (North American sample; Chiu et al., 2013), and D. melanogaster – SRR1738161. Graph-based clustering of NGS reads was performed using RepeatExplorer (Novák et al., 2013) on the Galaxy-based web server, following the pipeline described by Silva et al. (2016). This analysis provided genome-wide estimates of TE content and the relative contribution of different TE superfamilies, including Helitrons, which were then compared with the proportion of TE and Helitron copies overlapping CYP genes.

Statistical analysis

We first compared the size distributions of CYP genes and non-CYP genes within each species. For this purpose, a dataset of 500 additional genes was randomly selected from each genome. For D. suzukii, random genes were sampled using BEDTools v2.27.0 (Quinlan, 2014), and their orthologs in D. melanogaster were subsequently identified. Median gene lengths were used instead of means due to the asymmetrical distribution of gene sizes. Within each species, CYP gene lengths were compared with the lengths of the 500 randomly selected genes using the Mann-Whitney non-parametric test.

To account for differences in overall genome size and gene-length distributions between species, we normalized CYP gene lengths by the median size of the 500 randomly selected genes in each species. Normalized gene size was calculated as the length of each gene (in base pairs) divided by the species-specific median length of the 500 randomly selected genes. A Wilcoxon signed-rank test was then applied to compare normalized CYP sizes between orthologous CYPs of D. suzukii and D. melanogaster.

TE enrichment was compared between D. suzukii and D. melanogaster using four approaches: (1) the frequency of TEs within genes, including their flanking regions; (2) the frequency of TEs within CYP genes and flanking regions; (3) the frequency of TEs in CYP genes versus its frequency in other genes in the genome; and (4) the frequency of Helitron insertions in CYP genes versus its frequency in non CYP genes. The comparisons were performed using Chi-square tests with 1 degree of freedom and Yates’ continuity correction, which is usual in these cases. All annotated genes and intergenic regions in each genome were considered for these comparisons. Statistical analyses were conducted using SPSS^® version 18, and a significance threshold of p ≤ 0.05 was applied.

Results and discussion

Overall prevalence of TE insertions in CYP genes

Among the 76 CYP genes annotated for D. suzukii, 42 contained transposon sequences within or near the genes (Figure 1; Table 1; Supplementary Tables S1, S2). In D. melanogaster, 41 of the 91 genes analyzed harbored such insertions (Figure 1; Table 1; Supplementary Tables S3, S4). Although D. suzukii has fewer CYP genes overall, we detected a higher number of TE fragments in these genes (140 vs. 136). Most of this difference is mainly attributable to a markedly higher number of Helitron elements in D. suzukii (118 fragments; 84% of all TE insertions, Table 1) compared with D. melanogaster (32 fragments; 24%, Table 1). Other TE subclasses also differed in proportion between species (Table 1), with D. melanogaster showing 51% of LTR retrotransposons (69 fragments). Nevertheless, the absolute difference in total TE fragment counts between the two species is primarily explained by the excess of Helitron insertions in D. suzukii (Figure 1; Table 1).

Figure 1

Figure 1. Number of CYP genes with fragments of transposable elements.

Table 1

Table 1. Transposable element fragments belonging to subclasses and orders in CYP genes and flanking regions.

In flanking regions, TEs were detected both upstream and downstream of CYP genes, and were also detected within introns in both species (Figure 2). D. suzukii displayed more TE insertions in the 5′ flanking regions, where most promoter sequences are located. In contrast, D. melanogaster showed more TE insertions within introns, mainly due to a single gene, CYP307a2, which harbors 30 TE fragments in its intronic regions (Supplementary Table S3). In this gene, retroelements predominate across all regions (5′, 3′, and introns). The D. suzukii ortholog of CYP307a2 contains seven intronic insertions (Supplementary Table S1), where DNA transposons were the most abundant in all regions of this gene (5′, 3′, and introns). No TE insertions were detected within annotated exons.

Figure 2

Figure 2. Insertion position (5’ and 3’-flanking region, and intron region) of transposable elements in CYP genes of Drosophila suzukii and Drosophila melanogaster.

When comparing CYP genes with TE insertions in D. suzukii and D. melanogaster, we observed that some D. suzukii genes were longer than their orthologs in D. melanogaster (Figures 3A–J). Specifically, ten of the 36 D. suzukii genes containing TE insertions had additional exons and introns, organized as repetitive conserved blocks, compared with their D. melanogaster counterparts. In general, gene organization is highly conserved among species of the same order; therefore, exon and intron annotations are well supported and consistent with the phylogenetic relationships of the species (Rewitz et al., 2007). To further explore this pattern, we also examined two sister species of D. suzukii with available genome sequences, D. biarmipes and D. takahashii. In all four species, exon and intron annotations are confirmed at the transcript level (Chiu et al., 2013; Graveley et al., 2010; Drosophila biarmipes Annotation Release 101; Drosophila takahashii Annotation Release 101).

Figure 3

Figure 3. (A–J) Comparative analysis showing similarity between the CYP genes in Drosophila species. The intensity of red boxes between genes highlights the closest sequence above for which genes are denoted. Triangles represent the multiple transposable element insertions and their orientation. The phylogeny on the left is the phylogeny from Chiu et al. (2013), which was inferred by maximum-likelihood methodology. Genes are scaled to real length, except for flanking regions. Dmel, Drosophila melanogaster; Dtak, Drosophila takahashii; Dsuz, Drosophila suzukii; Dbia, Drosophila biarmipes.

Helitron elements contribute to CYP gene length variation

Helitron insertions account for only a small proportion of the total length of each longer CYP gene in D. suzukii, ranging from 0.43% to 5.63%, except CYP4e2, which contains 1,403 bp of Helitron sequence in a gene of 7,998 bp (17.54%) (Supplementary Table S1). These results indicate that, although Helitrons are consistently present in longer genes, they represent only a minor contribution to overall gene length. This suggests that Helitrons may have contributed to gene lengthening through rearrangements such as exon shuffling rather than by adding new sequences. In this view, Helitron activity could have facilitated structural reorganization of CYP genes, consistent with their known role in mediating exon capture and recombination events.

Analysis with genoPlotR, which compares gene and genome maps, revealed conserved exon structures across orthologous genes in D. suzukii, D. biarmipes, D. takahashii, and D. melanogaster (Figures 3A–J). The genes CYP12a4, CYP12e1, CYP6a18, CYP6a20, CYP6a21, CYP6a23, CYP6d5, and CYP4e2 of D. suzukii contain at least one Helitron fragment in the intron region.

The CYP4e2 gene in D. suzukii is larger than its D. melanogaster ortholog but contains only one fewer exon compared with the D. biarmipes ortholog (Figure 3H). Interestingly, even with one fewer exon, the D. suzukii CYP4e2 remains longer than its D. biarmipes counterpart. A Helitron fragment located between exons six and seven is present in D. suzukii but absent from D. biarmipes, suggesting that the loss of this exon in D. suzukii may have resulted from the Helitron insertion.

In contrast, the CYP4c3 gene in D. suzukii contains two Helitron insertions in the 3′ flanking region in a positive orientation (5’-3’) (Figure 3I). Its sister species D. takahashii carries a single Helitron insertion between exons five and six in a negative orientation (3’-5’). The structural differences observed in D. suzukii CYP4c3 could be explained by the presence of these two Helitrons and the rolling-circle recombination mechanism associated with this TE superfamily, whereby exons nine to eleven may have arisen through exon shuffling involving exons six to eight.

Unlike what was previously observed in D. suzukii, the CYP12a4 and CYP12e1 orthologs in D. biarmipes and D. takahashii lack transposon insertions (Figures 3A, B). However, in D. melanogaster, the CYP12a4 ortholog contains the BARI element in the 3’ flanking region (Figure 3A), as previously annotated (Bogwitz et al., 2005). The biological functions of CYP12a4 and CYP6a20 have been reported as insecticide responses and aggressive behavior, respectively - two key traits that contribute to the ecological success and invasive potential of insects.

Little or no similarity was observed for the CYP6w1 gene annotated in scaffold 2 (Figure 3J). However, the same gene annotated on scaffold 8 showed high similarity to its orthologs. BLAST searches performed at NCBI showed high sequence identity with the CYP6d2 genes of the sister species D. biarmipes (89%) and D. takahashii (87%). The D. suzukii CYP6d2 gene is absent from Gbrowser (SpottedWingFlyBase, 2013) but is predicted by the NCBI genome browser. On the other hand, the CYP6d5 gene is annotated in two scaffolds (99 and 1273) (Figure 3G). Both paralogs display high similarity to each other and the D. biarmipes ortholog, suggesting a duplication event in D. suzukii that may have been facilitated by Helitron-mediated insertion and transposition.

To determine whether the observed genomic size increase and TE insertions were specific to CYP genes in D. suzukii or also occurred in other gene families, we randomly selected 500 additional genes from each genome for comparison. We visually inspected 124 genes that were longer in D. suzukii than in their D. melanogaster orthologs. Among these longer D. suzukii genes, 45 carried a total of 249 Helitron copies, whereas in D. melanogaster, 41 genes carried 110 Helitron copies (Supplementary Table S7).

To allow for a fair comparison not influenced by the overall genome size differences between species, we compared CYP gene lengths with the median length of 500 randomly selected genes within each species. In D. melanogaster, CYPs were significantly smaller (median = 2,117 bp) than random genes (median = 5,603 bp; p = 0.025, Mann-Whitney). In D. suzukii, however, CYPs (median = 8,032 bp) did not differ significantly in size from random genes (median = 6,325 bp; p = 0.526).

Because CYP size differences could be influenced by overall genome size rather than TE-mediated arrangements, we normalized CYP lengths to the median size of the 500 random genes from each species. After normalization, CYPs in D. melanogaster were proportionally shorter (median ratio = 0.38) than in D. suzukii (median ratio = 1.27; p = 0.002, Wilcoxon). These results indicate that, on average, CYP genes in D. melanogaster are 38% shorter than the typical genes in its genome, whereas in D. suzukii they are 27% longer. Therefore, CYPs remain proportionally larger in D. suzukii than in D. melanogaster, even after normalization for genome size differences. This normalization was necessary to account for differences in overall genome size and structure between species, allowing a direct evolutionary comparison of relative gene length rather than absolute values, which can be biased by genome expansion or contraction.

Transposons are enriched in putative TFBS

Transposable elements (TEs) often carry transcription-factor binding sites (TFBS), and these sequences are preferentially retained within genes because they can contribute to transcriptional regulation (Jordan et al., 2003; Feschotte, 2008). Such retention likely reflects a byproduct of TE transposition combined with the host-level selection. We therefore searched for putative TFBS in all sequences identified within the CYP genes of D. suzukii (88 TFBS) and D. melanogaster (140 TFBS) (Figure 4; Supplementary Table S8). Of these, 35 of the 88 TFBS in D. suzukii were located within Helitrons, whereas only 5 of the 140 TFBS in D. melanogaster were found in Helitrons. Across all TE fragments located within CYP genes and their flanking regions in D. suzukii, 51 out of 140 (36%) carried at least one predicted TFBS (Supplementary Table S8). Although no explicit false-positive control or background frequency analysis was implemented, confidence scores for each predicted TFBS are provided in Supplementary Table S8.

Figure 4

Figure 4. Putative TFBS predicted for transposable elements inserted in CYP genes of Drosophila suzukii and Drosophila melanogaster.

As different classes of TEs were present in the CYP genes (Table 1), a broad diversity of TFBS motifs was expected (Thornburg et al., 2006). However, we detected little variation in the TFBS classes across TE families (Figure 4 and Supplementary Table S8). Although D. suzukii has a larger overall proportion of TE sequences in its genome (35.94%) compared with D. melanogaster (15.96%) (Table 2), the highest number of TFBS was found in TE fragments located within CYP genes of D. melanogaster (Supplementary Table S8). This is likely explained by the greater total base pair coverage of TEs within CYP genes and their flanking regions in D. melanogaster (76,813 bp; Supplementary Table S3) compared with D. suzukii (47,421 bp; Supplementary Table S1). It is important to note that these base pair values refer only to TE content in CYP genes and should not be confused with the genome-wide TE proportions reported in Table 2.

Table 2

Table 2. Genomic TE content in Drosophila suzukii and Drosophila melanogaster.

For both species, the putative TFBS Hunchback and CF2-II (Chorion factor 2) are consistently overrepresented (Figure 4). These proteins belong to the C2H2 zinc finger class of transcription factors. Hunchback is strongly expressed early in development (Nüsslein-Volhard and Wieschaus, 1980; Lehmann, 1988), whereas CF2-II is expressed later in embryogenesis (Shea et al., 1990). TE insertions in flanking regions and introns of CYP genes may therefore influence expression by harboring putative TFBSs. In this study, flanking regions were defined as ±10 kb upstream and downstream of each gene, encompassing proximal regulatory zones where transposon-derived enhancers or silencers are most likely to act. This, in turn, suggests that TEs may play an important role in facilitating adaptation to different environments in both species (Jordan et al., 2003; Feschotte, 2008; Shea et al., 1990; Thornburg et al., 2006). However, these regulatory implications remain hypothetical. Future functional and transcriptomic experiments will be required to confirm whether these TE-associated TFBSs have effects on CYP gene regulation or adaptive phenotypes.

TE content in Drosophila genomes

To place these findings in a genomic context, we next examined overall TE content in the genomes of D. suzukii and D. melanogaster. Approximately 36% of the assembled D. suzukii genome consists of TE sequences, compared with 16% in the genome of D. melanogaster (Table 2).

Three genome assemblies are currently available for D. suzukii. The first two were generated from North American samples (SRA096061; Chiu et al., 2013) – the same genomic data used in the present study – and from European samples (ERP001893; Ometto et al., 2013), both sequenced on the Illumina HiSeq2000 platform. Chiu et al. (2013) used automated homology comparison against 6,003 D. melanogaster TEs. In contrast, Ometto et al. (2013) applied a homology-based approach with RepeatMasker and the Repbase Insect library, estimating ~11% TE content in D. suzukii and ~17% in D. melanogaster. A third, near-chromosome-level assembly was produced by Paris et al. (2020) using PacBio long-read sequencing, which revealed that ~35% of the D. suzukii genome consists of repetitive sequences. However, the relative contribution of TE superfamilies was not determined. Differences among these studies likely reflect not only the methodologies used but also sequencing technology, which can affect the assembly completeness and thus TE content estimates.

In this study, we used RepeatExplorer, which integrates two complementary strategies for TE annotation: (1) homology-based searches against the Repbase library, and (2) de novo clustering to identify repetitive structures and patterns in the genome. This combined approach provides broader coverage, whereas earlier studies relying solely on homology-based annotation likely underestimated TE content. Supporting this explanation, Sessegolo et al. (2016), using the de novo pipeline dnaPipeTE (Goubert et al., 2015), estimated TE content at ~31% for D. suzukii and ~12% for D. melanogaster - values comparable to our results (~36% and ~16%, respectively). The primary difference is technical: dnaPipeTE requires local installation, while RepeatExplorer is web-based, but both rely on similar principles.

In the two species (Table 2), retrotransposons are the most abundant TE class, consistent with previous evidence that class I elements predominate in Drosophila genomes (Drosophila 12 Genomes Consortium, 2007). Among DNA transposons, Helitrons are the most abundant in both species and represent the second-largest TE category in D. suzukii. Actual TE content may be even higher, since RepeatExplorer tends to detect medium-to-high copy number and relatively recent TE insertions. In contrast, older, more diverged copies may not pass through similarity filters. Nevertheless, the clustering approach makes RepeatExplorer a fast and effective tool for initial TE analysis of Illumina data (Novák et al., 2013).

Comparing TE distribution between genes and intergenic regions revealed striking interspecific differences. In D. suzukii, TEs were found in 8.6% (10044 in 116977) of all annotated genes, whereas in D. melanogaster, 41.6% (35013 in 84181) of genes contained at least one TE insertion (p < 0.001). Moreover, in D. suzukii, 1.0% (103) of all TE copies and 1.0% (68) of all Helitron copies in the genome were located within CYP genes, while in D. melanogaster these proportions were 0.2% (87 TE copies) and 0.2% (19 Helitron copies), respectively. These differences in TE and Helitron proportions within CYP genes were statistically significant (p < 0.001).

Helitron distribution also differs between species. In D. suzukii, 95.6% of Helitrons are located in intergenic regions, whereas in D. melanogaster, 85.9% occur within genes. It should be noted that our methodology does not distinguish between complete Helitron elements and fragmented copies, which may contribute to differences observed between species. Nonetheless, fragmented copies likely represent remnants of once-intact Helitrons that were active earlier in the evolutionary history of these species. For this reason, our interpretations regarding the structural influence of Helitrons refer to their historical activity rather than current transposition or exon-shuffling events. This pattern may reflect a species-specific distribution of Helitrons, suggesting potential differences in TE dynamics, and does not alter the conclusion that D. suzukii harbors proportionally more Helitrons in intergenic regions.

Helitron elements and CYP gene evolution in D. suzukii

Metabolic resistance mediated by cytochrome P450 monooxygenases (CYPs) is an important adaptive trait in many insect species (Scott, 1999) and a common mechanism by which insects develop resistance to pesticides (Feyereisen, 1999). Transposable elements (TEs) are often found within or near resistance genes, providing indirect evidence of their involvement in the generation of adaptive genome changes (Catania et al., 2004; Chen and Li, 2007; Chung et al., 2007; Carareto et al., 2013; Casacuberta and González, 2013). Barbara McClintock (1984) first proposed that TE activation in response to stress could induce mutations that help organisms adapt to new environmental conditions.

In this study, we examined TEs associated with CYP genes in the highly invasive D. suzukii genome. We documented CYPs with varying TE contents, including TEs carrying putative transcription-factor binding sites (TFBS) and structural changes potentially mediated by rolling-circle transposons of the Helitron superfamily. We also found that the D. suzukii genome contains roughly twice the TE content of D. melanogaster, with Helitrons representing the most abundant subclass of class II DNA transposons in both species (Table 2).

In all CYP genes analyzed, TE fragments were located exclusively in flanking regions and introns, which is consistent with the view that TEs are generally tolerated in non-coding regions. However, TE insertions near genes can also create new regulatory networks (Feschotte, 2008), and changes in a gene-regulation network are thought to be very important during adaptive evolution (Casacuberta and González, 2013). In D. suzukii, TE insertions occurred predominantly (88%) in the 5’ flanking regions of CYP genes. Previous studies have shown that TE insertions in 5’ untranslated regions confer insecticide resistance – for example, in Drosophila CYP6g1, where the upstream ACCORD retroelement carries specific transcriptional enhancers (Daborn et al., 2002; Chung et al., 2007; Schmidt et al., 2010). D. melanogaster and D. simulans CYP genes harbor multiple TE insertions, many from the Helitron superfamily, which also carries putative TFBS (Carareto et al., 2013; review in Thomas and Pritham, 2015). These findings are consistent with previous hypotheses proposing that TEs may be gradually co-opted for host gene regulation (Chung et al., 2007; Feschotte, 2008).

The acquisition of new cis-regulatory elements via TE insertions provides opportunities for adaptation to novel environmental challenges (Casacuberta and González, 2013). Several LTR retrotransposons containing cis-regulatory motifs are highly expressed in response to specific stimuli (Kumar and Bennetzen, 1999), and these motifs often match those required for the activation of stress-response genes (Grandbastien et al., 2005). In our dataset, TE fragments carried putative TFBS involved in fly development, including Hunchback (embryo patterning) and CF2-II (cell differentiation). This suggests that CYPs may be particularly permissive to TE insertions because such sequences can act as donors of transcriptional regulatory signals, potentially altering gene expression at different developmental stages. Similar TFBS have been reported in TE sequences in silico (Babu et al., 2006; Thornburg et al., 2006; Carareto et al., 2013). Thus, TEs carrying TFBS may influence gene regulation and contribute to adaptation in Drosophila (Feschotte, 2008).

Beyond regulatory effects, TEs can also mediate structural genomic changes such as insertions, excisions, retrotranspositions, and exon shuffling. These processes can lead to exonization or intronization of TE sequences, and in some cases to exaptation, where TE-derived sequences acquire new functional roles. If beneficial, such insertions can be retained in the host genome. Feyereisen (1999) proposed two mechanisms by which CYP genes can evolve insecticide resistance: (1) structural changes in specific CYPs, such as exon gain or loss, and (2) increased gene expression. Exon shuffling, as first proposed by Gilbert (1987), is one route by which novel exons can arise. In our study, ten CYP genes (Figures 3A–J) displayed structural changes involving conserved blocks of exon gain, each associated with at least one Helitron insertion.

Helitrons – subclass 2 of Class II DNA transposons (Wicker et al., 2007) – are known to mediate exon shuffling, transduplication, and the introduction of novel regulatory elements (Morgante et al., 2005; Pritham and Feschotte, 2007; Thomas et al., 2014). These elements transpose via a rolling-circle mechanism that displaces a single DNA strand. A loop is formed before cleavage and reintegration elsewhere in the genome. They have a remarkable ability to capture and duplicate gene segments, and their transposition can include flanking sequences (Kapitonov and Jurka, 2007). While Helitrons are well studied in plants – especially maize, where they have captured and redistributed numerous genes (Lal et al., 2009; Barbaglia et al., 2012) – their role in Drosophila remains less explored.

Repetitive elements within introns may act as recombination hotspots, thereby promoting exon shuffling (Gilbert, 1987). In maize, most Helitron copies have incorporated gene segments, facilitating their amplification and dispersal throughout the genome (Yang and Bennetzen, 2009). A striking example outside Drosophila comes from Palmer amaranth (Amaranthus palmeri), where Helitron-mediated amplification of the EPSPS gene cassette confers glyphosate resistance (Molin et al., 2017). Our observations in D. suzukii CYP genes are consistent with such a mechanism, suggesting that Helitron insertions are associated with increased gene length.

We propose a hypothetical example of Helitron-mediated gene capture in Figure 5, illustrating how, if a Helitron bypasses its termination signal, strand displacement could continue through adjacent gene regions until a new signal is encountered, capturing and mobilizing those sequences (Kapitonov and Jurka, 2007; Grabundzija et al., 2016). For instance, in CYP12a4 (Figure 3A) and CYP6a20 (Figure 3D), the intronic arrangement, orientation, and high sequence similarity of exons support the possibility of Helitron-mediated capture during transposition (Figure 5A). Further studies should aim to experimentally validate Helitron-mediated gene capture in D. suzukii through long-read sequencing and transcriptomic analyses to confirm the presence of chimeric transcripts.

Figure 5

Figure 5. Hypothetical exon shuffling by rolling-circle transposon: (A) a longer gene formed by Helitron during its transposition; (B) the hole of two Helitron copies rearranging due to the similarity in the sequences.

Previous studies (Li et al., 2007) have documented genomic alterations leading to CYP overexpression in insecticide resistance. Mishra et al. (2018) reported that several longer CYP genes – CYP6w1, CYP6a20, CYP6a21, CYP6d5 – were significantly upregulated under insecticide exposure in D. suzukii, with responses varying between populations. Functional assays, such as CRISPR/Cas9-mediated knockouts, will be important for testing whether Helitron insertions affect gene expression and adaptive traits of this pest species.

In our comparative analysis of closely related Drosophila species (D. suzukii, D. melanogaster, D. biarmipes, and D. takahashii), the ten longer CYP genes revealed a largely conserved exon–intron organization across species, aligned with phylogenetic relationships (Figures 3A–J). However, D. suzukii displayed Helitron insertions absent from its sister species, contributing to gene length variation (CYP4e2 and CYP6d5). Although D. suzukii has fewer CYP genes than does D. melanogaster, previous work shows that longer genes can generate greater functional novelty than large gene families (Grishkevich and Yanai, 2014), in part because gene length is positively correlated with the number of splice variants (Kopelman et al., 2005). Gene lengthening, often driven by TE insertions (Grishkevich and Yanai, 2014), may thus contribute to adaptive structural changes. Our findings suggest that Helitrons could be a vehicle for such changes in D. suzukii CYP genes through their combined transposition and recombination activities.

Conclusion

In the CYP gene family of D. suzukii, we identified variations in the length of ten genes. Transposon sequences were present in intronic regions as well as in upstream and downstream flanking regions, with the Helitron superfamily representing the most frequent TE insertion in these genes.

We further examined the genomic TE content of D. suzukii and D. melanogaster by combining next-generation sequencing (NGS) reads with graph-based clustering to estimate repeat content. This approach enabled us to assess the presence and distribution of TEs, including Helitrons, and to explore their potential association with longer CYP genes, putative transcription-factor binding sites (TFBS), and patterns of TE abundance in D. suzukii.

While Helitrons represent the most abundant subclass of class II (DNA transposons) at the genome level in both D. suzukii and D. melanogaster, class I (retrotransposons) remain overall more abundant than class II elements in both species. Within CYP genes, however, D. suzukii shows a striking enrichment of Helitron fragments (84%), whereas D. melanogaster contains only 24% Helitron fragments. Thus, although Helitrons are the dominant subclass among DNA transposons genome-wide, they are disproportionately represented within CYP genes of D. suzukii, suggesting a lineage-specific pattern of Helitron this species.

Our findings support the view that TEs can play a significant role in adaptation. We observed structural changes in CYP genes – such as exon gain and loss events – suggesting that TEs may influence both gene architecture and regulatory functions. Notably, Helitron elements were enriched in putative TFBS, potentially affecting gene expression in response to environmental pressures. Differences in TFBS composition between TE fragments of D. suzukii and D. melanogaster further suggest a role for TEs in species-specific regulatory adaptations.

Studying the role of the Helitron superfamily within a genomic context is essential for understanding the adaptive mechanism that may have contributed to the evolution and pest status of D. suzukii. Future research should investigate the timing of Helitron insertions relative to gene divergence events, which will help to clarify the evolutionary dynamics of this element in D. suzukii. Such investigations may also shed light on the genetic factors underlying the species’ successful colonization and insecticide resistance. Ultimately, this knowledge could advance our understanding of TE mobility, genome size evolution, and the genetic basis of adaptation – providing both theoretical insights and practical applications for pest management, including comparisons between native and invasive populations.

Taken together, our results highlight Helitrons as a major driver of structural and regulatory diversification in CYP genes of D. suzukii, potentially underpinning its rapid adaptation and invasive success.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement

The manuscript presents research on animals that do not require ethical approval for their study.

Author contributions

PB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. SC-J: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – review & editing, Validation. VV: Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing – review & editing. MD: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. CNPQ, CAPES.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2025.1664211/full#supplementary-material

Footnotes

1. ^ http://gitlab.com/inkscape/inkscape.

References

Abraham J., Zhang A., Angeli S., Abubeker S., Michel C., Feng Y., et al. (2015). Behavioral and antennal responses of Drosophila suzukii (Diptera: Drosophilidae) to volatiles from fruit extracts. Environ. Entomol 44, 356–367. doi: 10.1093/ee/nvv013

PubMed Abstract | Crossref Full Text | Google Scholar

Asplen M. K., Anfora G., Biondi A., Choi D., Chu D., Daane K. M., et al. (2015). Invasion biology of spotted wing Drosophila (Drosophila suzukii): a global perspective and future priorities. J. Pest Sci. 88, 469–494. doi: 10.1007/s10340-015-0681-z

Crossref Full Text | Google Scholar

Babu M. M., Iyer L. M., Balaji S., and Aravind L. (2006). The natural history of the WRKY-GCM1 zinc fingers and the relations between transcription factors and transposons. Nucleic Acids Res. 34, 6505–6520. doi: 10.1093/nar/gkl888

PubMed Abstract | Crossref Full Text | Google Scholar

Barbaglia A. M., Klusman K. M., Higgins J., Shaw J. R., Hannah L. C., and Lal S. K. (2012). Gene capture by Helitron transposons reshuffles the transcriptome of maize. Genetics 190, 965–975. doi: 10.1534/genetics.111.136176

PubMed Abstract | Crossref Full Text | Google Scholar

Bogwitz M. R., Chung H., Magoc L., Rigby S., Wong W., O'Keefe M., et al. (2005). CYP12a4 confers lufenuron resistance in a natural population of Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 102, 12807–12812. doi: 10.1073/pnas.0503709102

PubMed Abstract | Crossref Full Text | Google Scholar

Bryne J. C., Valen E., Tang M. E., Marstrand T., Winther O., Krogh A., et al. (2008). JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res. 36, 102–106. doi: 10.1093/nar/gkm955

PubMed Abstract | Crossref Full Text | Google Scholar

Carareto C. M. A., Hernandez E. H., and Vieira C. (2013). Genomic regions harboring insecticide resistance-associated CYP genes are enriched by transposable element fragments carrying putative transcription factor binding sites in two sibling Drosophila species. Gene 537, 93–99. doi: 10.1016/j.gene.2013.11.080

PubMed Abstract | Crossref Full Text | Google Scholar

Casacuberta E. and González J. (2013). The impact of transposable elements in environmental adaptation. Mol. Ecol. 22, 1503–1517. doi: 10.1111/mec.12170

PubMed Abstract | Crossref Full Text | Google Scholar

Catania F., Kauer M. O., Daborn P. J., Yen J. L., Ffrench-Constant R. H., and Schlotterer C. (2004). Worldwide survey of an Accord insertion and its association with DDT resistance in Drosophila melanogaster. Mol. Ecol. 13, 2491–2504. doi: 10.1111/j.1365-294X.2004.02263.x

PubMed Abstract | Crossref Full Text | Google Scholar

Chen S. and Li X. (2007). Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes. BMC Evol. Biol. 7, 1–13. doi: 10.1186/1471-2148-7-46

PubMed Abstract | Crossref Full Text | Google Scholar

Chiu J. C., Jiang X., Zhao L., Hamm C. A., Cridland J. M., Saelao P., et al. (2013). Genome of Drosophila suzukii, the spotted wing Drosophila. G3 (Bethesda) 3, 2257–2271. doi: 10.1534/g3.113.008185

PubMed Abstract | Crossref Full Text | Google Scholar

Chung H., Bogwitz M. R., McCart C., Andrianopoulos A., Ffrench-Constant R. H., Batterham P., et al. (2007). Cis-regulatory elements in the Accord retrotransposon result in tissue-specific expression of the Drosophila melanogaster insecticide resistance gene CYP6g1. Genetics 175, 1071–1077. doi: 10.1534/genetics.106.066597

PubMed Abstract | Crossref Full Text | Google Scholar

Cini A., Ioriatti C., and Anfora G. (2012). A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management. Bull. Insectol. 65, 149–160.

Google Scholar

ConSite Software. (2004). Available online at: http://consite.genereg.net/. (Accessed August 1, 2019)

Google Scholar

Daborn P. J., Yen J. L., Bogwitz M. R., Le Goff G., Feil E., Jeffers S., et al. (2002). A single P450 allele associated with insecticide resistance in Drosophila. Science 297, 2253–2256. doi: 10.1126/science.1074170

PubMed Abstract | Crossref Full Text | Google Scholar

Deprá M., Poppe J. L., De T. D. C., HJ S., and Valente V. L. S. (2014). The first records of the invasive pest Drosophila suzukii in the South American continent. J. Pest Sci. 87, 379–383. doi: 10.1007/s10340-014-0591-5

Crossref Full Text | Google Scholar

Dreves A., Walton V., and Fisher G. (2009). A new pest attacking healthy ripening fruit in Oregon. 1–6, OSU Extension CatalogEM8991.

Google Scholar

Drosophila 12 Genomes Consortium (2007). Evolution of genes and genomes on the Drosophila phylogeny. Nature 450, 203–218. doi: 10.1038/nature06341

PubMed Abstract | Crossref Full Text | Google Scholar

Elliott T. A. and Gregory T. R. (2015). Do larger genomes contain more diverse transposable elements? BMC Evolutionary Biol. 15, 69. doi: 10.1186/s12862-015-0339-8

PubMed Abstract | Crossref Full Text | Google Scholar

Escudero L. A., Voschi D., and Batllor L. (2012). Drosophila suzukii, una nueva plaga de los frutales Vol. 347 (Spain: Vida Rural), 18–22.

Google Scholar

Feschotte C. (2008). The contribution of transposable elements to the evolution of regulatory networks. Nat. Rev. Genet. 9, 397–405. doi: 10.1038/nrg2337

PubMed Abstract | Crossref Full Text | Google Scholar

Feyereisen R. (1999). Insect P450 enzymes. Annu. Rev. Entomol 44, 507–533. doi: 10.1146/annurev.ento.44.1.507

PubMed Abstract | Crossref Full Text | Google Scholar

Feyereisen R. (2005). “Insect cytochrome P450,” in Comprehensive Molecular Insect Science, vol. 4. (Elsevier, Oxford), 177.

Google Scholar

FlyBase. (1993). Available online at: http://flybase.org.(Accessed June 1, 2019)

Google Scholar

Gilbert W. (1987). The exon theory of genes. Cold Spring Harb. Symp Quant Biol. 52, 901–905. doi: 10.1101/SQB.1987.052.01.098

PubMed Abstract | Crossref Full Text | Google Scholar

Goubert C., Modolo L., Vieira C., Valiente-Moro C., Mavingui P., and Boulesteix M. (2015). De novo assembly and annotation of the Asian tiger mosquito (Aedes albopictus) repeatome with dnaPipeTE from raw genomic reads and comparative analysis with the yellow fever mosquito (Aedes aEgypti). Genome Biol. Evol. 7, 1192–1205. doi: 10.1093/gbe/evv050

PubMed Abstract | Crossref Full Text | Google Scholar

Grabundzija I., Messing S. A., Thomas J., Cosby R. L., Bilic I., Miskey C., et al. (2016). A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes. Nat. Commun. 7, 10716. doi: 10.1038/ncomms10716

PubMed Abstract | Crossref Full Text | Google Scholar

Grandbastien M. A., Audeon C., Bonnivard E., Casacuberta J. M., Chalhoub B., Costa A. P., et al. (2005). Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae. Cytogenet. Genome Res. 110, 229–241. doi: 10.1159/000084957

PubMed Abstract | Crossref Full Text | Google Scholar

Graveley B. R., Brooks A. N., Carlson J. W., Cherbas L., Choi J., Joseph W., et al. (2010). The D. melanogaster transcriptome: modENCODE RNA-Seq. Nature 471, 473–479. doi: 10.1038/nature09715

PubMed Abstract | Crossref Full Text | Google Scholar

Grishkevich V. and Yanai V. (2014). Gene length and expression level shape genomic novelties. Genome Res. 24, 1497–1503. doi: 10.1101/gr.169722.113

PubMed Abstract | Crossref Full Text | Google Scholar

Guy L., Kultima J. R., and Andersson S. G. E. (2010). GenoPlotR: comparative gene and genome visualization in R. Bioinformatics 26, 2334–2335. doi: 10.1093/bioinformatics/btq413

PubMed Abstract | Crossref Full Text | Google Scholar

Harrop T. W. R., Sztal T., Lumb C., Good R. T., Daborn P. J., Batterham P., et al. (2014). Evolutionary changes in gene expression, coding sequence, and copy-number at the CYP6g1 locus contribute to resistance to multiple insecticides in Drosophila. PloS One 9, e84879. doi: 10.1371/journal.pone.0084879

PubMed Abstract | Crossref Full Text | Google Scholar

Hauser M. (2011). A historic account of the invasion of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) in the continental United States, with remarks on their identification. Pest Manag Sci. 67, 1352–1357. doi: 10.1002/ps.2265

PubMed Abstract | Crossref Full Text | Google Scholar

Hu B., Deng Y., Lu T., Ren M., Liu K., Rao C., et al. (2025). Inhibition of transcriptional regulation of detoxification genes contributes to insecticide resistance management in Spodoptera exigua. Commun. Biol. 8, 128. doi: 10.1038/s42003-025-07560-8

PubMed Abstract | Crossref Full Text | Google Scholar

Jordan I. K., Rogozin I. B., Glazko G. V., and Koonin E. V. (2003). Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends Genet. 19, 68–72. doi: 10.1016/S0168-9525(02)00006-9

PubMed Abstract | Crossref Full Text | Google Scholar

Joshi N. K., Biddinger D. J., Demchak K., and Deppen A. (2014). First report of Zaprionus Indianus (Diptera: Drosophilidae) in commercial fruits and vegetables in Pennsylvania. J. Insect Sci. 14, 1–4. doi: 10.1093/jisesa/ieu121

PubMed Abstract | Crossref Full Text | Google Scholar

Jurka J., Kapitonov V. V., Pavlicek A., Klonowski P., Kohany O., and Walichiewicz J. (2005). Repbase Update, a database of eukaryotic repetitive elements. Cytogenet. Genome Res. 110, 462–467. doi: 10.1159/000084979

PubMed Abstract | Crossref Full Text | Google Scholar

Kaneshiro K. Y. (1983). Drosophila (Sophophora) suzukii (Matsumura). Proc. Hawaiian Entomological Soc. 24, 179.

Google Scholar

Kapitonov V. V. and Jurka J. (2007). Helitrons on a roll: eukaryotic rolling-circle transposons. Trends Genet. 23, 521–529. doi: 10.1016/j.tig.2007.08.004

PubMed Abstract | Crossref Full Text | Google Scholar

Kopelman N. M., Lancet D., and Yanai I. (2005). Alternative splicing and gene duplication are inversely correlated evolutionary mechanisms. Nat. Genet. 37, 588–589. doi: 10.1038/ng1575

PubMed Abstract | Crossref Full Text | Google Scholar

Kumar A. and Bennetzen J. L. (1999). Plant retrotransposons. Annu. Rev. Genet. 33, 479–532. doi: 10.1146/annurev.genet.33.1.479

PubMed Abstract | Crossref Full Text | Google Scholar

Lal S., Oetjens M., and Hannah L. C. (2009). Helitrons: Enigmatic abductors and mobilizers of host genome sequences. Plant Sci. 176, 181–186. doi: 10.1016/j.plantsci.2008.11.004

Crossref Full Text | Google Scholar

Lasa R. and Tadeo E. (2015). Invasive Drosophilid Pests Drosophila suzukii and Zaprionus Indianus (Diptera: Drosophilidae) in Veracruz, Mexico. Fla Entomol 98, 987–988. doi: 10.1653/024.098.0332

Crossref Full Text | Google Scholar

Leblanc L., Rubinoff D., and Montgomery S. L. (2009). New immigrant Drosophilidae in Hawaii, and a checklist of the established immigrant species. Proc. Hawaii Entomol Soc. 41, 121–127.

Google Scholar

Lee J. C., Dreves M. J., Cave A. M., Kawai S., Isaacs R., Miller J. C., et al. (2015). Infestation of wild and ornamental non-crop fruits by Drosophila suzukii (Diptera: Drosophilidae). Ann. Entomol Soc. Am. 108, 117–129. doi: 10.1093/aesa/sau014

Crossref Full Text | Google Scholar

Lehmann R. (1988). Phenotypic comparison between maternal and zygotic genes controlling the segmental pattern of the Drosophila embryo. Development 104, 17–27. doi: 10.1242/dev.104.Supplement.17

Crossref Full Text | Google Scholar

Li X., Schuler M. A., and Berenbaum M. R. (2007). Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu. Rev. Entomol. 52, 231–255. doi: 10.1146/annurev.ento.51.110104.151104

PubMed Abstract | Crossref Full Text | Google Scholar

Marsano R. M., Caizzi R., Moschetti R., and Junakovic N. (2005). Evidence for a functional interaction between the Bari1 transposable element and the cytochrome P450CYP12a4 gene in Drosophila melanogaster. Gene 357, 122–128. doi: 10.1016/j.gene.2005.06.005

PubMed Abstract | Crossref Full Text | Google Scholar

Matsumura B. (1931). 6000 illustrated insects of Japan-Empire. Toko Shoin, Tokyo 1964. (Accessed June 8, 1931).

Google Scholar

McClintock B. (1984). The significance of responses of the genome to challenge. Science 226, 792–801. doi: 10.1126/science.15739260

PubMed Abstract | Crossref Full Text | Google Scholar

McKenzie J. A. and Batterham P. (1994). The genetic, molecular and phenotypic consequences of selection for insecticide resistance. Trends Ecol. Evol. 9, 166–169. doi: 10.1016/0169-5347(94)90079-5

PubMed Abstract | Crossref Full Text | Google Scholar

Mishra R., Chiu J. C., Hua G., Tawari N. R., Adang M. J., and Sial A. A. (2018). High-throughput sequencing reveals Drosophila suzukii responses to insecticides. Insect Sci. 25, 928–945. doi: 10.1111/1744-7917.12498

PubMed Abstract | Crossref Full Text | Google Scholar

Molin W. T., Wright A. A., Lawton-Rauh A., and Saski C. A. (2017). The unique genomic landscape surrounding the EPSPS gene in glyphosate-resistant Amaranthus palmeri: a repetitive path to resistance. BMC Genomics 18, 1–16. doi: 10.1186/s12864-016-3336-4

PubMed Abstract | Crossref Full Text | Google Scholar

Morgante M., Brunner S., Pea G., Fengler K., Zuccolo A., and Rafalski A. (2005). Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize. Nat. Genet. 37, 997–1002. doi: 10.1038/ng1615

PubMed Abstract | Crossref Full Text | Google Scholar

Müller P., Warr E., Stevenson B. J., Pignatelli P. M., Morgan J. C., Steven A., et al. (2008). Field-caught permethrin-resistant Anopheles Gambiae overexpress CYP6P3, a P450 that metabolises pyrethroids. PloS Genet. 4, e1000286. doi: 10.1371/journal.pgen.1000286

PubMed Abstract | Crossref Full Text | Google Scholar

Novák P., Neumann P., Pech J., Steinhaisl J., and Macas J. (2013). RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads. Bioinformatics 29, 792–793. doi: 10.1093/bioinformatics/btt054

PubMed Abstract | Crossref Full Text | Google Scholar

Nüsslein-Volhard C. and Wieschaus E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature 287, 795–801. doi: 10.1038/287795a0

PubMed Abstract | Crossref Full Text | Google Scholar

Ometto L., Cestaro A., Ramasamy S., Grassi A., Revadi S., Siozios S., et al. (2013). Linking genomics and ecology to investigate the complex evolution of an invasive Drosophila pest. Genome Biol. Evol. 5, 745–757. doi: 10.1093/gbe/evt034

PubMed Abstract | Crossref Full Text | Google Scholar

Paris M., Boyer R., Jaenichen R., Wolf J., Karageorg M., Green J., et al. (2020). Near-chromosome level genome assembly of the fruit pest Drosophila suzukii using long-read sequencing. Sci. Rep. 10, 11227. doi: 10.1038/s41598-020-67373-z

PubMed Abstract | Crossref Full Text | Google Scholar

Pritham E. J. and Feschotte C. (2007). Massive amplification of rolling-circle transposons in the lineage of the bat Myotis lucifugus. Proc. Natl. Acad. Sci. U.S.A. 104, 1895–1900. doi: 10.1073/pnas.0609601104

PubMed Abstract | Crossref Full Text | Google Scholar

Puinean A. M., Foster S. P., Oliphant L., Denholm I., Field L. M., Millar N. S., et al. (2010). Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae. PloS Genet. 6, e1000999. doi: 10.1371/journal.pgen.1000999

PubMed Abstract | Crossref Full Text | Google Scholar

Quinlan A. R. (2014). BEDTools: the Swiss-army tool for genome feature analysis. Curr. Protoc. Bioinf. 47, 11.12.1–34. doi: 10.1002/0471250953.bi1112s47

PubMed Abstract | Crossref Full Text | Google Scholar

RepeatMasker Open-4.0 Software. (1996). Available online at: http://www.repeatmasker.org (Accessed July 1, 2019).

Google Scholar

Rewitz K. F., O’Connor M. B., and Gilbert L. I. (2007). Molecular evolution of the insect Halloween family of cytochrome P450s: Phylogeny, gene organization, and functional conservation. Insect Biochem. Mol. Biol. 37, 741–753. doi: 10.1016/j.ibmb.2007.02.012

PubMed Abstract | Crossref Full Text | Google Scholar

Rota-Stabelli O., Blaxter M., and Anfora G. (2013). Drosophila suzukii. Curr. Biol. 23, R8–R9. doi: 10.1016/j.cub.2012.11.021

PubMed Abstract | Crossref Full Text | Google Scholar

Schlenke T. A. and Begun D. J. (2004). Strong selective sweep associated with a transposon insertion in Drosophila simulans. Proc. Natl. Acad. Sci. U.S.A. 101, 1626–1631. doi: 10.1073/pnas.0303793101

PubMed Abstract | Crossref Full Text | Google Scholar

Schmidt J. M., Good R. T., Appleton B., Sherrard J., Raymant G. C., Bogwitz M. R., et al. (2010). Copy number variation and transposable elements feature in recent, ongoing adaptation at the CYP6g1 Locus. PloS Genet. 6, 1–11. doi: 10.1371/journal.pgen.1000998

PubMed Abstract | Crossref Full Text | Google Scholar

Scott J. G. (1999). Cytochromes P450 and insecticide resistance. Insect Biochem. Mol. Biol. 29, 757–777. doi: 10.1016/S0965-1748(99)00038-7

PubMed Abstract | Crossref Full Text | Google Scholar

Sessegolo C., Burlet N., and Haudry A. (2016). Strong phylogenetic inertia on genome size and transposable element content among 29 species of flies. Biol. Lett. 12, 20160407. doi: 10.1098/rsbl.2016.0407

PubMed Abstract | Crossref Full Text | Google Scholar

Shea M. J., King D. L., Conboy M. J., Mariani B. D., and Kafatos F. C. K. (1990). Proteins that bind to Drosophila chorion cis-regulatory elements: A new C2H2 zinc finger protein and a C2C2 steroid receptor-like component. Genes Dev. 4, 1128–1140. doi: 10.1101/gad.4.7.1128

PubMed Abstract | Crossref Full Text | Google Scholar

Silva A. F., Dezordi F. Z., and Wallau G. L. (2016). Manual para caracterização genômica e análise evolutiva de elementos transponíveis utilizando diretamente reads de sequenciadores de alto desempenho Vol. 28 (Ribeirão Preto, SP: SBG).

Google Scholar

SpottedWingFlyBase. (2013). Available online at: http://spottedwingflybase.org. (Accessed June 14, 2019)

Google Scholar

Thomas J., Phillips C. D., Baker R. J., and Pritham E. J. (2014). Rolling-circle transposons catalyze genomic innovation in a mammalian lineage. Genome Biol. Evol. 6, 2595–2610. doi: 10.1093/gbe/evu204

PubMed Abstract | Crossref Full Text | Google Scholar

Thomas J. and Pritham E. J. (2015). Helitrons, the eukaryotic rolling-circle transposable elements. Microbiol. Spectr. 3(4). doi: 10.1128/microbiolspec.MDNA3-0049-2014

PubMed Abstract | Crossref Full Text | Google Scholar

Thornburg B. G., Gotea V., and Makałowski W. (2006). Transposable elements are a significant source of transcription-regulating signals. Gene 365, 104–110. doi: 10.1016/j.gene.2005.09.036

PubMed Abstract | Crossref Full Text | Google Scholar

Timmeren S. V. and Isaacs R. (2013). Control of spotted wing Drosophila, Drosophila suzukii, by specific insecticides and by conventional and organic crop protection programs. Crop Prot 54, 126–133. doi: 10.1016/j.cropro.2013.08.003

Crossref Full Text | Google Scholar

Walsh D. B., Bolda M. P., Goodhue R. E., Dreves A. J., Lee J., Bruck D. J., et al. (2011). Drosophila suzukii (Diptera: Drosophilidae): Invasive pest of ripening soft fruit expanding its geographic range and damage potential. J. Integr. Pest Manag 2, 1–7. doi: 10.1603/IPM10010

Crossref Full Text | Google Scholar

Wicker T., Sabot F., Hua-Van A., Bennetzen J. L., Capy P., Chalhoub B., et al. (2007). A unified classification system for eukaryotic transposable elements. Nat. Rev. Genet. 8, 973–982. doi: 10.1038/nrg2165

PubMed Abstract | Crossref Full Text | Google Scholar

Yang L. and Bennetzen J. L. (2009). Distribution, diversity, evolution, and survival of Helitrons in the maize genome. Proc. Natl. Acad. Sci. U.S.A. 106, 19922–19927. doi: 10.1073/pnas.0908008106

PubMed Abstract | Crossref Full Text | Google Scholar

Zhu F., Parthasarathy R., Bai H., Woithe K., Kaussmann M., Nauen R., et al. (2010). A brain-specific cytochrome P450 responsible for the majority of deltamethrin resistance in the QTC279 strain of Tribolium castaneum. Proc. Natl. Acad. Sci. U.S.A. 107, 8557–8562. doi: 10.1073/pnas.1000059107

PubMed Abstract | Crossref Full Text | Google Scholar